Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation

Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. Conclusion Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.


Introduction
Mouse embryonic stem cells (ESCs) are pluripotent cells that were initially isolated from blastocysts and cultured on cell cycle arrested mouse embryonic fibroblast (MEF) feeder cells using fetal calf serum (FCS) (1). Then, MEF and FCS were later replaced with leukemia inhibitory factor (LIF) (2) and bone morphogenetic protein 4 (BMP4) respectively (3). Later, the ground-state hypothesis of pluripotency by Smith and colleagues suggested that the chemical inhibition of endogenous differentiation signals, fibroblast growth factor 4 (FGF4) using PD0325901 and glycogen synthase kinase 3 (GSK3), with CHIR99021, known as 2i, can maintain cultured ESCs in the pluripotent state (4).
In another approach to the preserve ground state, Hassani et al. (5,6), reported that dual inhibition of mitogenactivated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors with PD0325901 and SB431542, known as R2i, results in highly-efficient generation of mouse ESCs even from refractory strains and single blastomeres (7). This medium also supports the efficient establishment of embryonic germ cell (EGC) lines from the primordial germ cells of mice (8) and rats (9).
In contrast to when the multifunctional GSK3 protein is inhibited in 2i, under R2i, the ESCs show better homogeneity (i.e. cell-to-cell conformity in expression of pluripotency genes such as Nanog and Dppa3), genomic integrity, and ground-state pluripotency. In which a less complex condition is required for investigating the molecular mechanisms of ground-state pluripotency (5,6,10).
These advantages of R2i prompted us to further assess the molecular mechanisms that underlie the transition from inner cell mass (ICM) to ESC. Recently, Totonchi et al. (11), reported the key genes involved in the transition from ICM to ESC via temporal microarray gene expression analysis. They also used deep hairpin bisulfite sequencing (DHBS) to show the methylation of individual CpG sites for three classes of repetitive elements, micro satellites (mSats), the 5´ untranslated region of L1Md_Tf (L1), and a class of LTR-retrotransposons (IAP-LTR1) (11,12). Their results indicated that DNA methyltransferases play a pivotal role in efficient ESC generation. However, the exact molecular mechanisms through which the derivation of ESCs takes place still needed to be clarified.
Here, we assessed the expression of key genes involved in pluripotency, epigenetic and early differentiation using quantitative real time-polymerase chain reaction (qRT-PCR) Then, the DNA methylation status of cytosine guanine dinucleotides (CpG) upstream of the transcription starting site of two pluripotency-related genes (Oct4 and Nanog) was determined using bisulfite genomic sequencing. Data was collected at different time points during the transition from ICM to ESC in R2i and compared to results from the serum culture condition.
The isolated ICMs, ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture in three independent replicates and stored at -80 o C. Each experimental group included 20 to 30 embryos/ outgrowths. All experiments were approved by the Ethical Committee of Royan Institute.

RNA isolation and quantitative real time-PCR
Total RNA was extracted from the three independent replicates using RNeasy micro kit (Qiagen, USA). The purity and concentration of the RNA was assessed and quantified by measuring the absorbance A260 nm/ A280 nm using a Biowave II spectrophotometer (WPA, Biochrom, UK). The quality and integrity of the total RNA was verified by electrophoresis. A total amount of 2 µg of total RNA was converted into cDNA using RevertAid cDNA synthesis kit and Random hexamer primers (Thermo Fisher Scientific, USA).
Quantitative real time PCR was carried out using SYBR Green master mix (ABI, Step one plus, USA). Primers (Table S1) (See Supplementary Online Information at www.celljournal.org) were designed by Perlprimer software and checked in Gene Runner software (http:// www.generunner.com). The reactions were carried out in triplicates and qRT-PCR amplification was performed using the following program; stage1: 95 o C for 10 minutes, stage 2 (40 cycles): 95 o C for 10 seconds, 60 o C for 60 seconds. The results were normalized against the reference gene (Gapdh) and compared with ICM. The relative quantification of gene expression was calculated using the ΔΔCt method.

DNA methylation assay
Pure DNA (1 µg) was treated with EpiTect Bisulfite Kit (Qiagen, USA). Semi-nested methylation specific primers (MSP) were designed for 2 promoter regions of two pluripotency-related genes (Nanog and Oct4), using Methprimer software. The primers used were: The PCR cycling program started at 95 o C for 5 minutes, then 32 cycles of 95 o C for 35 seconds, 53-54 o C for 40 seconds, and 72 o C for 35 seconds, followed by 72 o C for 10 minutes. Subsequently, 1 µl of bisulfite-treated DNA from each sample was amplified by AmpliTaq Gold kit (Life technology, USA). The PCR products were cloned using a TA-cloning kit (Invitrogen, USA). Next, 15 single white colonies were selected and the cloned fragments were amplified with M13 universal primers. The PCR product of each selected clone was analyzed by BiQ Analyzer software.

Statistical analysis
The data were analyzed by one-way analysis of variance (ANOVA) test, followed by a Tukey post-hoc test for determination of significant differences among groups and are presented as mean ± SD. Differences among groups were considered statistically significant at P<0.05.

Temporal expression of pluripotency and differentiation-specific genes during transition from inner cell mass to embryonic stem cells
Whole zona pellucida-free blastocysts were plated onto mitotically inactivated MEF feeder layer in R2i and serum conditions. The blastocyst-outgrowths in R2i culture have a typical compact morphology as opposed to those cultured in the serum medium. Also, it seems the number of trophectodermlike cells around attached blastocyst outgrowths decreased in R2i compared to serum (Fig.1). Next, to assess the temporal expression of key pluripotency-related genes during the procedure of mouse ESC establishment, we gently isolated the ICM outgrowths with a Pasture pipette on days 3, 5, and 7 after seeding the blastocysts. Then, qRT-PCR was performedto measure the expression of pluripotency markers namely, Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin as well as early differentiation markers, Gata6 as a primitive endoderm marker, Lefty2 as a primitive mesoderm marker and Cdx2 as a trophectoderm lineage marker (Fig.2).
R2i caused a significantly higher expression of pluripotencyrelated genes during ESC derivation, while in serum, the expression of these genes in outgrowths was not detected or was at very low levels. We observed two distinct expression patterns for the genes in R2i codition. In the first group, the expression continuously increased during derivation (Oct4 and Dax1), while Nanog, Sox2, Nodal, Dppa3 Tcf3, Rex1, Utf1, and β-Catenin were upregulated until day 5 and downregulated afterward. In addition, the early lineage differentiation genes were expressed at lower levels under the R2i condition compared to serum (P<0.001, Fig.2A).
Hierarchical clustering and heatmap analysis showed that the expression of most pluripotency-related genes was increased in R2i compared to the ICM and the highest level of gene expression was observed on day 5 (Fig.2B).

DNA methylation status of Oct4 and Nanog promoters and the expression of epigenetic-associated genes during embryonic stem cells derivation
Bisulfite sequencing was used to evaluate the methylation status of the twelfth and tenth CpGs in the promoter regions of the pluripotency-associated genes, Oct4 and Nanog respectively. Based on our data, the promoters of these genes were highly unmethylated during the transition from ICM to ESC in R2i condition whereas CpG dinucleotides of the regions in outgrowths were highly methylated in serum condition (Fig.3). These findings indicate that these promoters might be more active under R2i.
On the other hand, relative expression of epigenetic-related genes (Tet1, Carm1 and Setdb1) showed a significant upregulation under R2i (P<0.001, Fig.4). Notably, the expression of these genes was upregulated in a similar manner to that of the pluripotency-related genes in day 5.

Efficient embryonic stem cells generation after 5 days of treatment with R2i
As early evidence of high expression levels of pluripotency-association genes and hypomethylated DNA was found in R2i, we sought to determine whether ESCs could be established if we cultured ICM in R2i for 5 days and then continued the remainder of the culture in serum (5 days -R2i/serum). Concordantly, 3 groups of zona-free blastocysts (NMRI strain) were cultured on feeder cells (15 embryos for each group); entirely in R2i culture medium, 5 days in R2i and then serum and also solely in the serum condition. On day 7, the individual ICM outgrowths were picked from the outgrown trophectoderm using a Pasteur pipette and subsequently trypsinized (trypsin/EDTA, 0.05% w/v) and replated on freshly seeded MEF in 24-well plates. After 5 days, typical packed domed ESC-like colonies could be identified. The efficiency in the generation of ESCs in R2i was 100% and ~ 94% for 5 days-R2i/serum while in the serum condition, ESC colonies did not appear (Fig.5A). The ESCs passaged easily and showed dome-shaped colony morphologies (Fig.5B), high nuclear/cytoplasmic ratios, the ability to propagate following trypsin digestion and clonal growth from single cells while also displaying high levels of alkaline phosphatase activity (Fig.5C) and Oct4 expression. Therefore, 5 days treatment of whole blastocysts with R2i on MEF is sufficient for efficient generation of ESCs.

Discussion
In this study, we reported the gene expression and DNA methylation of ICM during ESC generation under dual inhibition of MEK and TGFβ signaling pathways with PD0325901 and SB431542 (known as R2i) that resulted in efficient generation of the ground-state pluripotency. R2i provides high genomic stability and an efficient transition from ICM to ESC (6). These advantages enable us to study the molecular mechanisms during ESC derivation. We analyzed the morphology of ICM-outgrowths on the MEF feeder layer in both, serum and R2i media. ICM-Outgrowths in serum had a larger fraction of trophoectoderm cells, while under R2i, the proliferation of these cells appeared to be inhibited. Therefore, we could conclude that ESC derivation on feeder cells in R2i medium reduces proliferation of trophoectoderm cells. In addition, the ICM-outgrowths were more compact and homogenous in comparison with the serum/LIF condition.
Next, we designed experiments to develop an appropriate strategy to explore genetic and epigenetic mechanisms that underlie ESC derivation. Here, we Temporal Epigenetic and Genetic Expression during ESC Derivation found that R2i significantly promotes upregulation of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and downregulates early differentiation genes (Gata6, Lefty2 and Cdx2). Previous studies have reported that increased expression of Dax1 in ESC, led to an increased expression of Oct4 (14,15). Likewise, Oct4 can bind to the promoter region of Dax1 and regulate its expression level (16). It has been shown that a balanced expression of Oct4, probably plays an important role in maintaining pluripotency (17). In addition, it was indicated that Gata6 and Cdx2 were downregulated during ICM outgrowth (18). Therefore, under the R2i condition, the ground-state of pluripotency during transition from ICM to ESC was maintained through the suppression of differentiationrelated pathways and enhancement of the expression of pluripotency-affiliated genes in ESCs (5)(6)(7)(8)(9)(10)(11)19).
Moreover, we found that the promoter regions of pluripotent-associated genes, Oct4 and Nanog, of ICM-outgrowths were significantly hypomethylated under R2i compared to the serum condition during the early days of ESC derivation. Moreover, we found that the genome of ESCs was hypermethylated in selected regions compared to ICM cells. Our data showed that DNA methylation status in ESCs is similar in relation to in line with the indings of a comparison between 2i and R2i (20,21). These patterns of DNA methylation have an essential role in the establishment of pluripotency under R2i (22,23). We found that inhibition of DNA methylation by a methyltransferase inhibitor, RG-108 resulted in efficient generation of ESCs under R2i condition (11).
The expression of epigenetic-related genes Tet1, Carm1 and Setdb1 was also significantly upregulated under the R2i condition. DNA methylation and the expression of epigenetic modifiers have been demonstrated in early embryo development and long-term maintenance of pluripotent cells (12,19,(24)(25)(26)(27). Previously, we had demonstrated that the expression of epigenetic-associated genes such as Dnmt3b, Dnmt3l, Chd8, Mtss1, Suz12, Eed, Wdr3, and Mat2b was significantly enhanced in the intermediate stages of ESC establishment (11). Finally, we clearly demonstrated that 5 days in the present of R2i with supporting MEF (5 days-R2i) is sufficient for efficient establishment of ESCs.

Conclusion
We demonstrate that establishment of ESCs requires upregulation of pluripotency-related genes and downregulation of differentiation-affiliated genes. Moreover, maintaining of DNA methylation at low levels established the ground-state pluripotency. In addition, we show the importance of the medium during the early days of ESC derivation which enables the capture of ESCs from blastocysts by maintaining the ground state of pluripotency.